THYMIDYLATE SYNTHASE REGULATION

胸苷酸合成酶（TS）的生物合成是近年来发现的

The biosynthesis of thymidylate synthase (TS) has recently been found to be down-regulated by binding of the protein to its own mRNA, and this regulation is involved in the development by cancer cells of resistance to several antimetabolites targeted at this enzyme. We are presently engaged in the physical characterization and quantification of this process. NCI-NMOB has observed that both the TS/mRNA binding and TS activity are strongly affected by the presence of mercaptoethanol (ME) and other reducing agents. We have hypothesized that both binding and activity are dependent on a reversible sulfhydryl switch, probably involving one or more cysteines near the active pocket of the enzyme. To test this hypothesis, we have constructed a kinetic model of such a process and examined it for its ability to account for experimental observation. The model allows for the binding of TS to two different mRNA locations chemically cleaved by T1-RNAse during assay: the reversible redox switch, an unknown amount of active TS introduced into the assay before addition of ME; and an ordered mechanism for synthesis of thymidine from uridylate and methylenetetrahydrofolate. We estimated a binding constant of 0.76 nM, a redox equilibrium constant of 4 x 10-9, and an initial TS active fraction of 1.1%. Most importantly, we found no significant difference between parameters estimated either from a fitting of combined binding and enzyme data sets or individual fittings, suggesting that the same reducing site is involved in both binding and active pocket locales. Furthermore,the calculated enzyme activity has been found to be in very close agreement with TS activity obtained from lactobacillus caseii.